Time-Domain Lifetime Standards

The need foi Ufeliine standuds for time-domain measurements has been recognized for some time. A number of labontories have suggested samples as single-decay-time standards. The data ate typically repotted ouly in tables Clhbles n.3-n.S). so representative figures are not 

Flpiro ii.9. RcfNeoaitithe Inqiiency-domaUi intensi dec. Flom Ref. S. 

Thompson, R. Bn rad Grftttoa,E. 1988. Phase floorometiic method for detamniatioa of staodsid lifetimes, AiioL Chem. 80 670-674. 

Castelli, F., 1985, Detennlnatum of comet refnence fliMcescenee lifeliraei by self-consistent internal calitenkm, Rev. Sd. Instrum. 56 538-542. 

HuoiescencelifetuneieiiBteane Standards fw the range ai89 to 115 naooaecaode, AtuaL Blodiem. 57 593-604. 

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Implementation of time domain FLIM methods is comparatively straightforward in laser scanning microscopes (LSMs). Here, pointscanning is used so that single channel lifetime detection suffices. In principle, standard fluorescence lifetime detection equipment developed for spectroscopy can be used in combination with point-scanning systems and a pulsed laser. 

In frequency-domain FLIM, the optics and detection system (MCP image intensifier and slow scan CCD camera) are similar to that of time-domain FLIM, except for the light source, which consists of a CW laser and an acousto-optical modulator instead of a pulsed laser. The principle of lifetime measurement is the same as that described in Chapter 6 (Section 6.2.3.1). The phase shift and modulation depth are measured relative to a known fluorescence standard or to scattering of the excitation light. There are two possible modes of detection heterodyne and homodyne detection. 

J. R. Lakowicz, 1. Gryczynski, G. Laczko, and D. Gloyna, Picosecond fluorescence lifetime standards for frequency- and time-domain fluorescence,./. Fluorescence 1, 87-93 (1991). 

An electronic or vibrational excited state has a finite global lifetime and its de-excitation, when it is not metastable, is very fast compared to the standard measurement time conditions. Dedicated lifetime measurements are a part of spectroscopy known as time domain spectroscopy. One of the methods is based on the existence of pulsed lasers that can deliver radiation beams of very short duration and adjustable repetition rates. The frequency of the radiation pulse of these lasers, tuned to the frequency of a discrete transition, as in a free-electron laser (FEL), can be used to determine the lifetime of the excited state of the transition in a pump-probe experiment. In this method, a pump energy pulse produces a transient transmission dip of the sample at the transition frequency due to saturation. The evolution of this dip with time is probed by a low-intensity pulse at the same frequency, as a function of the delay between the pump and probe pulses.1 When the decay is exponential, the slope of the decay of the transmission dip as a function of the delay, plotted in a log-linear scale, provides a value of the lifetime of the excited state. 

If the spectral properties of the molecules are too similar for spectral identification, they may have different fluorescence lifetimes such that they could be identified in the time domain. By using pulsed laser excitation and time-correlated single-photon counting, which is the standard method for measuring fluorescence lifetimes [47], the researchers at Los Alamos have... 

It is valuable to have fluorophores of known lifetimes for use as lifetime standards in time-domain oc frequency-domain measurements. Perhaps more inportaot than the actual lifetime is kno edge that the fluoroi icw disfda single-expcmential decays. Such fluoro c es are useful for testing the time-resolved instruments for systonatic errors. We have summarized the results on lifetime standards from several laboratm ies. Th e is no attempt to compare the values or to evaluate which values are more reliable. Much of the data is from our laboratory because it was readily available with all the exp imen-tal details. 

Boens N, Qin WW, Basaric N et al (2007) Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy. Anal Chem 79 2137-2149... 

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